Method for providing assistance in determining fertility

Abstract

A method for providing assistance in determining the fertility of a female subject according to the present invention comprises a step for generating information for determining the fertility of the female subject by using the proportion of cells in which an X chromosome has been lost in somatic cells collected from the female subject.

Description

Methods for assisting in determining fertility

[0001] The present invention relates to a method for aiding in determining the fertility of a female subject.

[0002] In recent years, there has been an increase in couples who are having difficulty conceiving despite their desire to do so. Fertility (the ability to conceive) declines with age for both men and women. However, the rate at which fertility declines with age varies greatly from person to person. For women in particular, delays in pregnancy and childbirth significantly impact their life plans. Therefore, if a woman can identify her low fertility early on, she may be able to receive early infertility testing and treatment, potentially mitigating delays in pregnancy and childbirth.

[0003] There are various tests to find the cause of infertility (infertility tests), such as AMH test, hysterosalpingography, fallopian tube airflow test, and fallopian tube water flow test (see, for example, Non-Patent Document 1). On the other hand, there have been few reports on fertility tests.

[0004] Minoru Irahara and 24 others, "Reproductive Medicine Guidelines for Patients," [Online], February 8, 2023, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, [Retrieved August 30, 2024], Internet <URL: https: / / www.gynecology-htu.jp / reproduction / dl / seishokuiryo_gl.pdf>

[0005] As mentioned above, there are various infertility tests available, but few fertility (pregnancy) tests have been reported.

[0006] It is an object of the present invention to provide a method to aid in determining the fertility of a female subject.

[0007] The present invention relates to the following methods for assisting in determining fertility: [1] A method for assisting in determining the fertility of a female subject, comprising a step of generating information for determining the fertility of the female subject using a proportion of cells in which an X chromosome has been lost in somatic cells collected from the female subject. [2] A method for assisting in determining the fertility of a female subject, comprising a step of comparing the proportion of cells in which an X chromosome has been lost in somatic cells collected from the female subject with a predetermined first threshold. [3] The method for assisting in determining fertility according to [1] or [2], further comprising a step of measuring the proportion of cells in which the X chromosome has been lost in the somatic cells collected from the female subject. [4] The method for assisting in determining fertility according to [2], further comprising the step of comparing the proportion of cells in which the X chromosome has been lost in the somatic cells with a predetermined second threshold, wherein in the step of comparing the proportion of cells in which the X chromosome has been lost in the somatic cells with the first threshold, the proportion of cells in which the X chromosome has been lost is compared with the first threshold only for the female subject whose proportion of cells in which the X chromosome has been lost in the somatic cells is less than the second threshold. [5] The method for assisting in determining fertility according to [4], wherein the second threshold is within the range of 5.0 to 15.0%. [6] The method for assisting in determining fertility according to any one of [1] to [5], wherein the somatic cells are blood cells. [7] The method for assisting in determining fertility according to any one of [1] to [6], wherein the cells in which the X chromosome has been lost are XO cells.

[0008] According to the present invention, a method for assisting in determining the fertility of a female subject can be provided.

[0009] 1A and 1B are flowcharts of a method for assisting in determining fertility according to an embodiment of the present invention. FIG. 2 is a graph showing the measurement results of FAM and HEX fluorescence intensity for droplets derived from a single female subject. FIG. 3 is a graph showing the percentage of XO cells in general women and women visiting infertility clinics. FIG. 4 is a graph showing the percentage of XO cells in general women and women with infertility.

[0010] The method for assisting in determining fertility according to the present invention relates to a method for assisting medical professionals and the like in determining fertility by providing materials for determining the fertility of a female subject (information for determining the fertility of a female subject).

[0011] The phenomenon of acquired sex chromosome loss in hematopoietic cells is known. The loss of the Y chromosome is called mLOY (Mosaic loss of Chromosome Y), and the loss of the X chromosome is called mLOX (Mosaic loss of Chromosome X). Various methods have been proposed for detecting mLOY and mLOX, but all of them have problems in terms of sensitivity, cost, and complexity. The inventors have improved droplet PCR and found a method that can detect mLOY and mLOX on a cell-by-cell basis with high sensitivity and ease. Using this method, the inventors investigated the percentage of cells producing mLOX (LOX cells) in the blood cells of women and noticed a correlation between the percentage of LOX cells and fertility. Based on this finding, the inventors conducted diligent research and found that the percentage of cells with a missing X chromosome in the somatic cells of a woman can be used as a criterion for determining her fertility, thus completing the present invention.

[0012] In other words, the method for assisting in determining fertility according to the present invention includes a step of generating information for determining the fertility of a female subject using the percentage of cells in which the X chromosome is missing (hereinafter also referred to as the "percentage of LOX cells") collected from the female subject. The percentage of LOX cells may or may not be compared with a threshold.

[0013] For example, a method for assisting in determining fertility according to the present invention includes a step of comparing the proportion of cells in which an X chromosome has been lost (proportion of LOX cells) in somatic cells collected from a female subject with a predetermined first threshold value, where the "first threshold value" is a preset value that serves as a criterion for determining fertility.

[0014] The method for assisting fertility determination according to the present invention may further include, in addition to the above steps, the steps of measuring the percentage of cells lacking an X chromosome (the percentage of LOX cells) in somatic cells collected from a female subject, and / or comparing the percentage of cells lacking an X chromosome (the percentage of LOX cells) in somatic cells with a predetermined second threshold. Here, the "second threshold" is a value that is set in advance and serves as a criterion for determining whether or not a subject is suitable for fertility determination. These steps are not mandatory and may be performed optionally. These steps are performed before the step of comparing the percentage of LOX cells with a predetermined first threshold.

[0015] 1A and 1B are flow charts of a method for assisting in determining fertility according to an embodiment of the present invention.

[0016] As shown in FIG. 1A, a method for assisting in determining fertility according to one embodiment of the present invention includes a step (S110) of measuring the proportion of LOX cells in somatic cells collected from a female subject, and a step (S130) of comparing the proportion of LOX cells in the somatic cells with a predetermined first threshold value.

[0017] As shown in Figure 1B, another embodiment of the present invention provides a method for assisting in determining fertility, which includes the steps of: measuring the percentage of LOX cells in somatic cells collected from a female subject (S110); comparing the percentage of LOX cells in somatic cells with a predetermined second threshold (S120); and comparing the percentage of LOX cells in somatic cells with a predetermined first threshold (S130).

[0018] Each of the above steps will be described below. In this specification, a numerical range indicated by "to" means a numerical range including the numerical values ​​written before and after "to".

[0019] (1) Measurement of the proportion of LOX cells (S110) First, the proportion of LOX cells in somatic cells collected from female subjects is measured. As mentioned above, the step of measuring the proportion of LOX cells (S110) is not required. In other words, if the measurement result of the proportion of LOX cells is already available, it is not necessary to measure the proportion of LOX cells again.

[0020] The type of somatic cells that constitute the population is not particularly limited. Examples of somatic cells that constitute the population include blood cells (white blood cells), oral cells that can be collected by oral swabbing, fibroblasts such as those from skin, hair matrix cells, and epithelial cells in urine.

[0021] The number of missing X chromosomes in cells with a missing X chromosome (LOX cells) is not particularly limited. Examples of LOX cells include XO cells, which have one missing X chromosome; XXO cells, which are cells from patients with triple X syndrome and have one missing X chromosome; and XOO cells, which are cells from patients with triple X syndrome and have two missing X chromosomes. Since the X chromosome contains genes essential for survival, not all X chromosomes are usually lost. Therefore, the LOX cells that are usually measured are XO cells.

[0022] The method for detecting LOX cells is not particularly limited. Examples of methods for detecting LOX cells include droplet PCR, fluorescence in situ hybridization (FISH), karyotype analysis, DNA microarrays, and whole-genome sequencing (WGS). From the viewpoint of efficiently detecting LOX cells in a large number of cells, droplet PCR is preferred. In droplet PCR, for example, droplets are formed so that one cell is taken up by one droplet, and droplet PCR is performed so that PCR is stopped during the exponential amplification phase, and the signal intensity derived from the X chromosome is measured for each droplet, thereby allowing the number of X chromosomes in each cell to be measured sequentially (see International Publication No. 2022 / 138736). Below, as a specific example of this method, an example of detecting XO cells from blood cells (leukocytes) will be described.

[0023] (Blood pretreatment) Red blood cells are removed from the peripheral blood of women by hemolysis. Next, the remaining cells are pretreated to increase their membrane permeability.

[0024] (Preparation of PCR premix) A PCR premix is ​​prepared by mixing primers, probes, DNA polymerase, deoxynucleoside triphosphate (dNTPs), cofactors, surfactants, and buffer. The primers and probes are designed to target multiple locations on the X chromosome and multiple locations on a pair of autosomes (e.g., chromosome 2) for comparison. A first fluorescent dye (e.g., FAM) is used for probes targeting the X chromosome, and a second fluorescent dye (e.g., HEX) is used for probes targeting the comparison autosomes (e.g., chromosome 2).

[0025] (Droplet Preparation) The suspension of pretreated cells and the PCR premix are mixed and encapsulated in water-in-oil emulsion type droplets. A QX200 droplet generator (Bio-rad), for example, can be used to form the droplets. The cell concentration in the suspension is adjusted so that in most droplets, the number of cells in each droplet is either one or zero.

[0026] (PCR and Fluorescence Intensity Measurement) The prepared droplets are subjected to a thermal cycler for cell lysis and PCR. The number of PCR cycles is set so that PCR stops during the exponential amplification phase (see International Publication No. 2022 / 138736). After PCR is complete, the fluorescence intensity of the first fluorescent dye (e.g., FAM) and the second fluorescent dye (e.g., HEX) in each droplet is measured. For measuring fluorescence intensity, for example, a QX200 droplet reader (Bio-rad) can be used.

[0027] (Detection of XO cells) Figure 2 is a graph showing an example of the measurement results of the fluorescence intensity of the first fluorescent dye (here FAM) and the second fluorescent dye (here HEX) for each droplet derived from a single woman. The horizontal axis represents the intensity of the second fluorescent dye (HEX) corresponding to the number of targets on the autosome (here chromosome 2) being compared, and the vertical axis represents the intensity of the first fluorescent dye (FAM) corresponding to the number of targets on the X chromosome.

[0028] The number of targets on the autosome (chromosome 2) contained in each droplet corresponds to the fluorescence intensity of the measured second fluorescent dye (HEX). When a droplet contains one cell, one cell usually contains two autosomes, so the fluorescence intensity of the second fluorescent dye (HEX) in each droplet is approximately the same.

[0029] Similarly, the number of targets on the X chromosome contained in each droplet corresponds to the measured fluorescence intensity of the first fluorescent dye (FAM). When a droplet contains one cell, a normal female cell (XX cell) contains two X chromosomes, so the fluorescence intensity of the first fluorescent dye (FAM) in each droplet is approximately the same. However, in cells that are missing one X chromosome (XO cells), the fluorescence intensity of the first fluorescent dye (FAM) in each droplet is weaker because there is one less X chromosome (see Figure 2).

[0030] As described above, the fluorescence intensity of the first fluorescent dye (FAM) allows us to distinguish between signals from normal cells (XX cells) and signals from XO cells that have lost one X chromosome (see Figure 2). In Figure 2, "XX cells" shows clusters of signals from droplets containing one normal cell (XX cell), and "XO cells" shows clusters of signals from droplets containing one cell that has lost one X chromosome (XO cell). The signals scattered separately to the upper right of these clusters are signals from droplets containing two or more cells.

[0031] For each woman, the number of XX cell signals and XO cell signals are measured, and the proportion of XO cells is calculated using the following formula. Preferably, only the number of signals from droplets containing one cell is used as the number of signals for each cell type.

[0032] The number of somatic cells used to measure the percentage of LOX cells in a single female subject is not particularly limited and can be set according to the sensitivity of the measurement method. For example, the number of somatic cells used to measure is preferably 1,500 or more, and particularly preferably 3,000 or more. The larger the number of cells, the more accurately the percentage of LOX cells can be measured, and the more accurate the determination of fertility can be.

[0033] (2) Comparison with the second threshold (S120) Next, the percentage of LOX cells in somatic cells is compared with a predetermined second threshold. As mentioned above, the "second threshold" is a value that is set in advance and serves as a criterion for determining whether or not a subject is suitable to be judged for fertility. If the percentage of LOX cells is above the second threshold, it is possible that the measurement of the percentage of LOX cells was not performed accurately, or that the female subject has a major congenital or acquired abnormality, and therefore it may not be possible to properly determine fertility. For this reason, if the percentage of LOX cells is above the second threshold, the flow may be terminated without performing the next step of comparison with the first threshold (S130). If the percentage of LOX cells is below the second threshold, the process proceeds to the next step of comparison with the first threshold (S130). That is, in the next step of comparison with the first threshold (S130), the percentage of LOX cells may be compared with the first threshold only for female subjects whose percentage of LOX cells in somatic cells is below the second threshold (see Figure 1B).

[0034] The second threshold is not particularly limited and can be set arbitrarily. For example, the second threshold is in the range of 5.0 to 15.0%, and is 10.0%.

[0035] As mentioned above, the comparison step with the second threshold (S120) is not required. That is, for all female subjects whose percentage of LOX cells in somatic cells is known, the percentage of LOX cells may be compared with the first threshold (see Figure 1A).

[0036] (3) Comparison with First Threshold (S130) Next, the proportion of LOX cells in the somatic cells collected from the female subject is compared with a predetermined first threshold. As described above, the "first threshold" is a preset value that serves as a fertility assessment criterion. For example, if the proportion of LOX cells is less than the first threshold, fertility is not impaired, whereas if the proportion of LOX cells is equal to or greater than the first threshold, fertility is assessed as impaired.

[0037] The first threshold is not particularly limited and can be set arbitrarily. For example, the first threshold may be in the range of 0.5–4.0%, 0.5–3.0%, and 1.0%. Alternatively, multiple first thresholds may be set, and the percentage of LOX cells may be compared to multiple first thresholds.

[0038] The above procedure can provide a fertility assessment material (information for assessing the fertility of a female subject) for a female subject. Medical professionals and others can use this assessment material to assess the fertility of the female subject. Furthermore, the female subject can also understand her own fertility.

[0039] In the above example, the proportion of LOX cells in somatic cells is compared with a threshold value (first threshold value). However, information for determining the fertility of a female subject may be generated without comparing the proportion of LOX cells in somatic cells with a threshold value. For example, data on the proportion of LOX cells by age may be accumulated, and information may be generated indicating whether the proportion of LOX cells in a female subject aged X falls within (A) the confidence interval for the proportion of LOX cells in a population aged X - 8 or younger, (B) the confidence interval for the proportion of LOX cells in a population aged X - 7 or older and younger than X, (C) the confidence interval for the proportion of LOX cells in a population aged X or older and younger than X + 7, or (D) the confidence interval for the proportion of LOX cells in a population aged X + 8 or older. If the proportion of LOX cells in the female subject falls within (B), this serves as the criterion for determining that the fertility of the female subject is age-appropriate or older. Here, the confidence interval for the proportion of LOX cells in the population is not particularly limited, but may be, for example, the interquartile range of the proportion of LOX cells in the population (between the first and third quartiles).

[0040] Our research has shown that while a high proportion of LOX cells in somatic cells tends to decrease the success rate of natural conception, a high proportion of LOX cells in somatic cells does not decrease the success rate of in vitro fertilization (IVF). Therefore, even if a female subject has a high proportion of LOX cells in somatic cells, she can be encouraged to try IVF or other methods instead of giving up on pregnancy.

[0041] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0042] [Example 1] The percentage of cells lacking the X chromosome (XO cells) in the blood cells (white blood cells) of nine general women aged 20-35 (women who had never visited an infertility clinic, average age 28.9 years) and 24 women aged 20-35 who were visiting an infertility clinic (average age 31.9 years) was examined using the following procedure.

[0043] (Blood pretreatment) Red blood cells were removed from the peripheral blood of a woman by hemolysis. Next, the remaining cells were pretreated to increase their membrane permeability.

[0044] (Preparation of PCR Premix) A PCR premix was prepared by mixing primers, probes, DNA polymerase, deoxynucleoside triphosphate (dNTPs), cofactors, surfactants, and buffer. The primers and probes were designed to target multiple locations on the X chromosome and multiple locations on chromosome 2. FAM was used as the fluorescent dye for probes targeting the X chromosome, and HEX was used as the fluorescent dye for probes targeting chromosome 2.

[0045] (Droplet Preparation) The suspension of pretreated cells and the PCR premix were mixed and encapsulated in water-in-oil emulsion type droplets. A QX200 droplet generator (Bio-rad) was used to form the droplets. The cell concentration in the suspension was adjusted so that the number of cells in each droplet was either one or zero in most droplets.

[0046] (PCR and Fluorescence Intensity Measurement) The prepared droplets were placed in a thermal cycler to lyse the cells and perform PCR. The number of PCR cycles was set so that PCR stopped during the exponential amplification phase. After PCR was completed, the fluorescence intensity of FAM and HEX in each droplet was measured. Fluorescence intensity was measured using a QX200 droplet reader (Bio-Rad).

[0047] (Calculation of the Percentage of XO Cells) Figure 2 is a graph showing the measurement results of the FAM and HEX fluorescence intensities of droplets derived from a single woman. The horizontal axis represents the HEX intensity corresponding to the number of targets on chromosome 2, and the vertical axis represents the FAM intensity corresponding to the number of targets on the X chromosome. Signals from normal XX cells and LOX cells were distinguished from XO cells based on the FAM fluorescence intensity. In Figure 2, "XX cell" represents a cluster of signals from a droplet containing one normal cell (XX cell), and "XO cell" represents a cluster of signals from a droplet containing one cell (XO cell) that has lost one X chromosome. Note that the signals scattered above and to the right of these clusters represent signals from droplets containing two or more cells.

[0048] For each woman, the number of XX cell signals and XO cell signals were measured, and the proportion of XO cells was calculated using the following formula. For each cell type, only the number of signals from droplets containing one cell was used.

[0049] Figure 3 is a graph showing the percentage of XO cells in general women aged 20 to 35 (women who have never visited an infertility clinic, average age 28.9 years) and women aged 20 to 35 who visit an infertility clinic (average age 31.9 years). This graph suggests that there is a significant difference in the percentage of XO cells between general women and women who visit an infertility clinic, and that the percentage of XO cells corresponds to fertility (ability to become pregnant).

[0050] Example 2 The proportion of cells lacking the X chromosome (XO cells) in blood cells (leukocytes) was examined using the same procedure as in Example 1 for 123 general women (women who had never visited an infertility clinic, average age 34.0 years) and 381 infertile women (women who had not conceived for one year despite having unprotected sexual intercourse in the hope of becoming pregnant, average age 32.0 years).

[0051] Figure 4 is a graph showing the percentage of XO cells in 123 normal women (mean age 34.0 years) and 381 infertile women (mean age 32.0 years). This graph suggests that there is a significant difference in the percentage of XO cells between normal women and infertile women, and that the percentage of XO cells corresponds to fertility (fertility). In fact, logistic regression analysis was performed on 507 women after removing the effect of BMI. When women were divided into those with an XO cell percentage of less than 0.87% and those with an XO cell percentage of 0.87% or more, the probability of unsuccessful natural conception was 2.16 times higher for women in the latter group than for women in the former group. On the other hand, a Mann-Whitney U test was performed on 172 of the 381 infertile women who had undergone three or more IVF treatments. No significant difference was found in LOX levels between the successful and unsuccessful IVF groups.

[0052] This application claims priority based on Japanese Patent Application No. 2024-154718, filed September 9, 2024. The contents of the specification and drawings of that application are incorporated herein by reference in their entirety.

[0053] The method for assisting in determining fertility according to the present invention is useful, for example, as a fertility test.

Claims

1. A method for assisting in determining the fertility of a female subject, comprising a step of generating information for determining the fertility of the female subject using the proportion of cells in which an X chromosome has been lost in somatic cells collected from the female subject.

2. A method for assisting in determining the fertility of a female subject, comprising the step of comparing the proportion of cells in which an X chromosome has been lost in somatic cells collected from the female subject with a predetermined first threshold value.

3. A method for assisting in determining fertility according to claim 1 or claim 2, further comprising a step of measuring the proportion of cells in which the X chromosome has been lost in the somatic cells collected from the female subject.

4. The method for assisting in determining fertility according to claim 2, further comprising the step of comparing the proportion of cells in which the X chromosome has been lost in the somatic cells with a predetermined second threshold, wherein in the step of comparing the proportion of cells in which the X chromosome has been lost in the somatic cells with the first threshold, the proportion of cells in which the X chromosome has been lost is compared with the first threshold only for those female subjects whose proportion of cells in which the X chromosome has been lost in the somatic cells is less than the second threshold.

5. A method for assisting in determining fertility as described in claim 4, wherein the second threshold is within the range of 5.0 to 15.0%.

6. A method for assisting in determining fertility according to any one of claims 1 to 5, wherein the somatic cells are blood cells.

7. A method for assisting in determining fertility according to any one of claims 1 to 6, wherein the cells in which the X chromosome has been lost are XO cells.

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